Exploring parasite heterogeneity using single-cell RNA-seq reveals a gene signature among sexual stage Plasmodium falciparum parasites

Abstract

The malaria parasite has a complex lifecycle, including several events of differentiation and stage progression, while actively evading immunity in both its mosquito and human hosts. Important parasite gene expression and regulation during these events remain hidden in rare populations of cells. Here, we combine a capillary-based platform for cell isolation with single-cell RNA-sequencing to transcriptionally profile 165 single infected red blood cells (iRBCs) during the intra-erythrocytic developmental cycle (IDC). Unbiased analyses of single-cell data grouped the cells into eight transcriptional states during IDC. Interestingly, we uncovered a gene signature from the single iRBC analyses that can successfully discriminate between developing asexual and sexual stage parasites at cellular resolution, and we verify five, previously undefined, gametocyte stage specific genes. Moreover, we show the capacity of detecting expressed genes from the variable gene families in single parasites, despite the sparse nature of data. In total, the single parasite transcriptomics holds promise for molecular dissection of rare parasite phenotypes throughout the malaria lifecycle.

Single Cell RNA-sequencing reveals cellular heterogeneity, stage transition and antigenic variation during stress adaptation in synchronized Plasmodium falciparum

“…As per this study majority of parasites (13 out of 17 individual cells) show expression of one dominant var gene belonging to var sub group B. Furthermore, only a few parasites showed expression of two var genes per cells [3].…”

“…However, the mechanism of selection of a single var gene for expression and suppression of rest of the var genes is still not clear. Recent study using scRNA-seq employing Smart-seq2 has looked into the expression of var genes at the ring stage of the parasite [3]. As per this study majority of parasites (13 out of 17 individual cells) show expression of one dominant var gene belonging to var sub group B.…”

“…Thus, parasite maintains balance in transmission and persistent infection by an unknown mechanism. Recent single-cell RNA sequencing (scRNA-seq) suggests commitment to gametocytes during the preceding cycles by epigenetic regulation of master regulator of sexual development, AP2-G [2][3][4]. However, the environmental cues and the hitherto mechanism therein, which initiate the gametogenesis are not yet clear.…”

“…Plasmodium is known to have tight gene regulation orchestrated at multiple levels by chromatin structure [16,17], transcription [18][19][20] and translation [21,22], which helps in timely expression of different genes required at various stages of parasite growth [23]. Despite tight-gene regulation there is a huge amount of transcriptional heterogeneity exists in the parasite populations [3]. The cell-to-cell heterogeneity can be attributed to various environmental and physiological conditions such as availability of nutrients, temperature fluctuations, cell-cycle progression and different morphological states.…”

“…It provides information pertinent to different states co-exist in a cell population leading to dynamic cell-to-cell variability in mRNA numbers and concentrations. Earlier study in Plasmodium using scRNA-seq has revealed the heterogeneity in the parasite population during intraerythrocytic like cycle [3].…”